Plants have the ability to produce a diverse range of structures, including more than 20,000 different terpenoids, flavonoids, alkaloids, and fatty acids. Fatty acids have been extensively exploited for industrial uses in products such as lubricants, plasticizers, and surfactants. In fact, approximately one-third of vegetable oils produced in the world are already used for non-food purposes (Ohlrogge, J (1994) Plant Physiol. 104:821-26).
Most plant fatty acids are obtained from seed oils, which consists primarily of storage oil in the form of triglycerols, with minority contributions primarily from membrane lipids, which are predominantly phospholipids. Seed oils from different species of higher plants contain a total of more than 210 naturally occurring fatty acids, which differ by the number and arrangement of double or triple bonds and various functional groups, such as hydroxyls, ketones, epoxys, cyclopentenyl or cyclopropyl groups, furans or halogens (van de Loo et al. (1993) in Lipid Metabolism in Plants. (Ed: Moore T S Jr.; CRC Press, Boca Raton, Fla.) pp 91-126). These include at least 33 structurally distinct monohydroxylated plant fatty acids, and 12 different polyhydroxylated fatty acids have been described (reviewed by van de Loo et al. (1993) in Lipid Metabolism in Plants. (Ed: Moore T S Jr.; CRC Press, Boca Raton, Fla.) pp 91-126; Smith (1970) in Progress in the Chemistry of Fats and Other Lipids, Vol. 11 pt 1. (Ed: Holman, RT, Ed) p 139-177).
The most commonly occurring fatty acids in both membrane and storage lipids are 16- and 18-carbon fatty acids which may have from zero to three, methylene-interrupted, unsaturations. These are synthesized from the fully saturated species as the result of a series of sequential desaturations which usually begin at the DELTA 9 carbon and progress in the direction of the methyl carbon (Browse and Somerville (1991) Biochim Biophys Acta 1082(1): 1-26). Fatty acids which cannot be described by this simple algorithm are generally considered “unusual” even though several, such as lauric (12:0), erucic (22:1) and ricinoleic acid (12D-hydroxyoctadec-cis-9-enoic acid) are of significant commercial importance. The present invention is directed to the biosynthesis of hydroxylated fatty acids, such as ricinoleic acid in castor (Ricinus communis) seed.
Ricinoleic acid is synthesized by oleate-12-hydroxylase. A gene for this enzyme has been isolated from castor bean endosperm and sequenced (U.S. Pat. Nos. 6,028,248, 5,801,026, and 5,668,292). However, an enzymatic activity within a species frequently exists in different variant forms, such as isoenzymes or isoforms. Isoenzymes often possess different properties, such as tissue-specific distribution or differential distribution in different cellular compartments. Isoenzymes typically have the same general catalytic activity, but differ in physical properties, such as, optimum pH, isoelectric point and stability under differing in vitro or in vivo conditions. Different isoenzymes may also demonstrate differing characteristics in transgenic organisms, making them more or less effective for a specific purpose. Therefore, it is useful to isolate different isoforms of known enzymes, such as oleate-12-hydroxylase.